Proton and electron transfer during the reduction of molecular oxygen by fully reduced cytochrome c oxidase: a flow-flash investigation using optical multichannel detection

Proton and electron transfer events during the reaction of solubilized fully reduced bovine heart cytochrome c oxidase with molecular oxygen were investigated using the flow-flash technique. Time-resolved spectral changes resulting from ligand binding and electron transfer events were detected simultaneously with pH changes in the bulk. The kinetics and spectral changes in the visible region (450-750 nm) were probed by optical multichannel detection, allowing high spectral resolution on time scales from 50 ns to 50 ms. Experiments were carried out in the presence and absence of pH-sensitive dyes (carboxyfluorescein at pH 6.5, phenol red at pH 7.5, and m-cresol purple at pH 8.5) which permitted separation of spectral changes due to proton transfer from those caused by ligand binding and electron transfer. The transient spectra recorded in the absence of dye were analyzed by singular-value decomposition and multiexponential fitting. Five apparent lifetimes (0.93 microseconds, 10 microseconds, 36 microseconds, 90 microseconds, and 1.3 ms at pH 7.5) could consistently be distinguished and provided a basis for a reaction mechanism consistent with our most recent kinetic model Sucheta, A., Szundi, I., and Einarsdóttir, O. (1999) Biochemistry 37, 17905-17914]. The dye response indicated that proton uptake occurred concurrently with the two slowest electron transfer steps, in agreement with previous results based on single-wavelength detection Hallén, S., and Nilsson, T. (1992) Biochemistry 31, 11853-11859]. The stoichiometry of the proton uptake reactions was approximately 1.3 +/- 0.3, 1.4 +/- 0.3, and 1.6 +/- 0.5 protons per enzyme at pH 6.5, 7.5, and 8.5, respectively. The electron transfer between heme a and CuA was limited by proton uptake on a 90 microseconds time scale. We have established the lower limit of the true rate constant for the electron transfer between CuA and heme a to be approximately 2 x 10(5) s-1.